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SOMATIC HYBRIDIZATION

SOMATIC HYBRIDIZATION. Fundamentals of Biotechnology. This is a non conventional genetic procedure involving fusion b.w isolated protoplast under in vitro condition and subsequent development of their product ( heterokaryon ) to a hybrid plant Or

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SOMATIC HYBRIDIZATION

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  1. SOMATIC HYBRIDIZATION Fundamentals of Biotechnology

  2. This is a non conventional genetic procedure involving fusion b.w isolated protoplast under in vitro condition and subsequent development of their product (heterokaryon) to a hybrid plant Or Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties is called somatic hybridization

  3. Heterokaryon: binucleate cell (heterocyte) Hybrid: nuclei are fused (synkaryocyte) Hybridization Cybrid: fusion of nucleated and enucleated different somatic cell. (Cybridization)

  4. Somatic hybridization technique 1. isolation of protoplast from suitable plants 2. Fusion of the protoplasts of desired species/varieties 3. Identification and Selection of somatic hybrid cells 4. Culture of the hybrid cells 5. Regeneration of hybrid plants

  5. Isolation of Protoplast (Separartion of protoplasts from plant tissue) 1. Mechanical Method 2. Enzymatic Method A. Sequential (two step) B. Mixed (simultaneous)

  6. 1. Mechanical Method Plant Tissue CellsPlasmolysis MicroscopeObservation of cells Release of protoplasm Cutting cell wall with knife Collection of protoplasm

  7. 1. Mechanical Method • Used for vacuolated cells like onion bulb scale, radish and beet root tissues (storage tissues) • Low yield of protoplast • Laborious and tedious process • Low protoplast viability

  8. Enzymatic Method Leaf sterlization, removal of epidermis Mixed (simultaneous) Sequential (two step) Plasmolysed cells Plasmolysed cells Pectinase +cellulase Pectinase Protoplasm released Release of isolated cells Protoplasm released cellulase Isolated Protoplasm

  9. Enzymatic Method • Used forvariety of tissues and organs including leaves, petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores • Mesophyll tissue - most suitable source • High yield of protoplast • Easy to perform • More protoplast viability

  10. Protoplast Fusion (Fusion of protoplasts of two different genomes) 1. Spontaneous Fusion 2. Induced Fusion Intraspecific Intergeneric Chemofusion Mechanical Fusion Electrofusion

  11. Spontaneous Fusion • Protoplast fuse spontaneously during isolation process mainly due to physical contact • Intraspecific produce homokaryones

  12. Induced Fusion • Chemofusion- fusion induced by chemicals • Types of fusogens • PEG • NaNo3 • High pH/Ca 2+ ions • Polyvinyl alcohal

  13. Induced Fusion • Mechanical Fusion- Physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette

  14. Induced Fusion • Electrofusion- Fusion induced by electrical stimulation • Pearl chain of protoplasts is formed by low strength electric field (10kv m-1) • Fusion of protoplasts of pearl chain is induced by the application of high strength electric field (100kv m-1) for few microseco • High voltage pulse induce a reversible breakdown of plasma membrane at the sit of cell contact, leading to fusion and consequently membrane reorganization. • Simple, quicker and more efficient than chemical induced fusion.

  15. Cell Wall Regeneration May be complete in two to several days Although protoplast in culture generally start regenerating a cell wall within a few hours after isolation. Protoplast lost their characteristic spherical shape once the wall formation is complete. Regeration of cell wall can be demonstrated using Calcalfluor White ST fluoresecent Stain (USA) or Tinapol solution (UK)

  16. Identification and Selection of somatic hybrid cells • Hybrid identification- Based on difference between the parental cells and hybrid cell with respect to • Pigmentation • Cytoplasmic markers • Fluorochromes like FITC (fluorosceinisothiocyanate) and RITC (Rhodamineisothiocyanate) are used for labelling of hybrid cells • Presence of chloroplast • Nuclear staining • Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-orcein stain

  17. Hybrid Selection (Several markers are used ) • Genetic complementation • Phytotoxins • Specific amino acid • Auxin autotrophy • Antibiotics • Auxotrophic and metabolic mutants • Chromosomal analysis • Herbicides

  18. Culture of the hybrid cells Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions.

  19. Regeneration of hybrid plants • Plants are induced to regenerate from hybrid calli • These hybrid plants must be at least partially fertile, in addition to having some useful property, to be of any use in breeding schemes.

  20. Advantages of somatic hybridization • Production of novel interspecific and intergenic hybrid • Pomato (Hybrid of potato and tomato) • Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids • Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters

  21. Advantages of somatic hybridization • Production of heterozygous lines in the single species which cannot be propagated by vegetative means • Studies on the fate of plasma genes • Production of unique hybrids of nucleus and cytoplasm

  22. Limitations of Somatic hybridization • Poor regeneration of hybrid plants • Non-viability of fused products • Not successful in all plants. • Production of unfavorable hybrids • Lack of an efficient method for selection of hybrids • No confirmation of expression of particular trait in somatic hybrids

  23. Micropropagation

  24. Introduction In nature plants propagate either Sexually (seeds generation) results heterogeneity Or Asexually (vegetative multiplication) produce genetically identical plants. Multiplication of genetically identical copies of a cultivar by asexual reproduction is called clonal propagation. Via tissue culture called micropropagation,

  25. What is Micropropagation? “… the asexual or vegetative propagation (multiplication) of plants in vitro “ Implies - regeneration - multiplication - uniformity ??

  26. Basic in vitro propagation ...

  27. Micropropagation (contin) • Positives and negatives of micropropagation • positives • rapid multiplication rates • low space requirement • negatives • labor costs • high overhead (equipment, facilities, supplies) • loss by contamination • danger of variation

  28. STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Shoot elongation 5. Root induction / formation 6. Acclimatization

  29. STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Shoot elongation 5. Root induction / formation 6. Acclimatization

  30. Tip bud Starting material formicropropagation Leaf Axillary bud Internode Root

  31. Selection of plant material ... • Part of plant • Genotype • Physiological condition • Season • Position on plant • Size of explant

  32. Physiological state - of stock plant • Vegetative / Floral • Juvenile / Mature • Dormant / Active • Carbohydrates • Nutrients • Hormones

  33. STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Shoot elongation 5. Root induction / formation 6. Acclimatization

  34. Disinfestation • Stock plant preparation • Washing in water • Disinfecting solution • Internal contaminants • Screening

  35. The medium • Minerals • Sugar • Organic ‘growth factors’ • Growth regulators • Gelling agent • Other additives

  36. Physical Environment • Temperature • Moisture • Light

  37. STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Shoot elongation 5. Root induction / formation 6. Acclimatization

  38. Origins of new shoots ... • Terminal extension • Lateral / Axillary buds • Adventitious (de novo, re-differentiation) • Callus differentiation

  39. auxins cytokinins gibberelic acid ethylene abscisic acid Role of growth regulators ... • Cell division • Differentiation • Cell expansion • Apical dominance

  40. STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Shoot elongation 5. Root induction / formation 6. Acclimatization

  41. Shoot elongation ... • Basal ‘hormone free’ medium • Gibberellins • Carry-over of hormones

  42. STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Elongation 5. Root induction / formation 6. Acclimatization

  43. Root initiation ... • Auxins • Co-factors • C : N ratio • Light / darkness • Initiation vs growth • Juvenility / rejuvenation • Genotype

  44. STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Elongation 5. Root induction / formation 6. Acclimatization

  45. Acclimatization (hardening) - survival of the new plant when removed from the in vitro environment - will be covered later.

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